This project is aimed at developing a new class of drugs, which act by inhibiting pathogenic activities of senescent cells. Cell senescence occurring as a result of aging or cellular damage, is associated with upregulation and secretion of proteins implicated in many age-related diseases, including cancer, Alzheimer's disease, atherosclerosis and arthritis. These proteins are induced in response to the expression of cell cycle inhibitors of cyclin-dependent kinase inhibitor (CKI) family, such as p21Waf1/Cip1 or p16Ink4A. Senex Biotechnology has identified a novel class of drug-like small molecules, SNX2-class compounds, which reduce the induction of disease-associated genes, senescence-associated morphological changes and secretion of tumor- promoting factors by CKI-arrested cells. Phase I studies of this project revealed that SNX2-class compounds affect CKI interaction with cyclin/CDK complexes in such a way that CDK2 kinase activity becomes much more susceptible to the inhibition by CKI, whereas transcriptional activation by CKI is greatly diminished. Thus SNX2-class compounds both prevent the upregulation of disease-associated proteins and enhance the tumor- suppressive activity of CKIs. The first aim of the Phase II program is to define the target of SNX2-class compounds at the biochemical level, as an approach to target-based design of drugs with the same activity. To test a hypothesis that SNX2-class compounds act by affecting post-translational modifications of the protein components of CKI/cyclin/CDK complexes, the component proteins will be analyzed for changes in their modifications upon treatment with SNX2-class compounds. Once the protein modifications susceptible to SNX2-class compounds are identified, the corresponding modifying enzymes will be tested as targets for inhibition by these compounds in cell-free assays. In another line of analysis, components of p21-containing protein complexes, the binding of which is affected by SNX2-class compounds, will be identified. The potential role of such proteins in CKI-induced transcription will be tested by RNA interference assays. The second aim of the program is to carry out chemical optimization and structure-activity analysis of lead compounds of SNX2 family. The optimization strategy, based on the activity of 40 SNX2-related compounds, will guide the synthesis of chemical derivatives, which will be tested for efficacy in preventing the induction of transcription by p21 and for their effect on the target enzyme activity, as identified in the first aim. These assays will elucidate the structure-activity relationship of SNX2 family proteins for designing more efficacious compounds of the same family. The third aim of the program is to test lead compounds of SNX2 family for acute toxicity in mice and for ex vivo and in vivo efficacy in inhibiting the tumor-promoting activity of senescent fibroblasts. These studies will provide an in vivo proof-of-principle for therapeutic use of SNX2-class compounds. Successful completion of Phase II will allow us to generate strategies and candidate compounds for preclinical and clinical development of drugs with a novel mechanism of action and with broad applicability to cancer and Alzheimer's disease. PUBLIC HEALTH RELEVANCE: With the aging of the American population, chronic age-related diseases, such as Alzheimer's disease, atherosclerosis, chronic renal disease and cancer, are becoming an increasingly greater burden on the society. There is a great need for developing novel pharmaceuticals that would be efficient against these largely incurable diseases. Senex Biotechnology, Inc. is a small drug discovery company that targets a newly discovered biological regulatory pathway, which causes increased production of proteins implicated in many age-related diseases. Senex has discovered prototype drug-like small molecules that block this disease- promoting pathway. The goal of this Phase II SBIR application is to identify the molecular target of these compounds, increase their biological activity and demonstrate their efficacy in an animal model. These studies will make it possible to develop this new class of molecules as potential drugs against the diseases of old age, including cancer and Alzheimer's disease.